Ozone-induced gene expression in plants

ABSTRACT

The present invention relates to new DNA sequences, a method for producing new plants which contain a new DNA sequence, the coding sequence thereof being expressed after ozone induction. The invention also relates to said new plants and the use of DNA sequences to produce ozone-responsive gene expression in plants and plant cells. Moreover, it relates to a new promoter, the specificity thereof being increased by removal of the ozone response capacity thereof.

This invention relates to new DNA sequences, a method for producing newplants which contain a new DNA sequence, the coding sequence thereofbeing expressed after ozone induction. The invention also relates tosaid new plants and the use of DNA sequences to produce ozone-responsivegene expression in plants and plant cells. Moreover, it relates to a newpromotor, the specificity of which is increased by removal of the ozoneresponse capacity thereof.

The ozone concentrations in the lower troposphere above the continentsof the northern hemisphere have steadily increased over the past onehundred years as a result of greater industrial activities (Volz andKley (1988) Nature 332, 240-242). Meanwhile, ozone values reachintermittent peak concentrations of 100 nL/L up to nL/L in Europe andNorth America (Krupa et al. (1995) Environ. Pollut: 87, 119-126).

The phytotoxicity of the air pollutant ozone has been well tested anddocumented, e.g., in Heagle (1989) Annu. Rev. Phytopathol. 27, 397-423;Heath (1994) in: Alscher, Wellburn (ed) “Plant responses to the gaseousenvironment”, pp. 121-145, Chapman & Hall, London. A decreased netphotosynthesis and an increased early senescence are usually the resultof such ozone impact which, consequently, results in diminished plantgrowth and a lower harvest yield.

Although ozone penetrates the plant cell through open stomata by meansof diffusion, the ozone concentration in the intercellular spaces of theleaf is almost zero, irrespective of the environmental ozoneconcentration (Laisk et al. (1989) Plant Physiol. 90, 1163-1167). It iscurrently assumed that ozone reacts quickly with components of the cellwalls and the plasmalemma and is converted into reactive oxygen species,such as peroxide-anions, hydroxyl radicals and hydrogen peroxide whichwere detected in ozone-treated plant material by using electron spinresonance spectroscopy (Mehlhorn et al. (1990) Physiologia Plantarum 79,377-383). The so-called “oxidative burst”, i.e. the fast development ofa relatively high quantity of reactive oxygen species, can lead to adramatic disturbance of the normal cell function due to alteration ofthe permeability of the plant membrane, inactivation of redox-sensitiveproteins and increased lipid peroxidation.

Recent tests conducted on ozone-treated plants showed an increasedbiosynthesis of non-specific, defensive enzymes, the function of whichis to protect live cells against damage due to oxidative stress(Kangasjärvi et al. (1994 Plant, Cell and Environment 17, 783-794). Yetthe signal-transduction chain, which is responsible for theozone-induced gene activation, which transmits to the cell core therelevant information about the formation of apoplastic, reactive oxygenspecies, has not been understood up to now. Various factors, such as theincrease of calcium concentration in cytosol (Price et al. (1994) ThePlant Cell 6, 1301-1310), the formation of salicylic acid (Klessig andMalamy (1994) Plant Mol. Biol. 26, 1439-1458) and the phytohormonejasmon acid (Farmer (1994) Plant Mol. Bio. 26 1423-1437) and ethylene(Ecker (1995) Science 268, 667-674) are currently being discussed aspossible signal connections, caused by oxidative stress, which generallyplay a part in defence reactions of plants.

Tests conducted on ozone-gassed tobacco plants showed that ozone causesan increased expression of various disease-resistant genes, namely a fewPR-(pathogenesis-related) proteins (Ernst et al. (1992) Plant Mol. Biol.20, 673-682; Ernst et al. (1996) J. Plant Physiol. 148, 215-221;Eckey-Kaltenbach et al. (1994) Plant Physiol. 104, 67-74). These resultsindicate that oxidative stress, caused by ozone, influences theexpression and regulation of defensive genes of plants in a similar wayas that described regarding pathogenic attack. Only very limitedinformation on cis-regulatory elements and transcription factors, whichpossibly play a part in the control of gene expression of non-specificdefensive genes as a response to various environmental influences, isavailable at present (Lee et al. (1994) Eur. J. Biochem. 226, 109-114).However, based on previous results, it can be assumed that with respectto the genes coding for PR-proteins, separate or at least only partlyoverlapping ways of signal transduction exist (Somssich (1994) in: Nover(ed) “Plant promoters and transcription factors”, pp. 163-179, SpringerPublishing House, Berlin; Dolferus et al. (1994) Plant Physiol. 105,1075-1087).

With respect to the activity of the stilbene synthase (STS), which takespart in the phytoalexin synthesis, it is known that in adult plants itis induced by environmental stress factors, such as, e.g., pathogenicattack (Langcake (1981) Physiol. Plant Pathol. 18, 213-226), ultravioletlight (Fritzemeier and Kindl (1981) Planta 151, 48-52) and ozone(Rosemann et al. (1991) Plant Physiol. 97, 1280-1286. Contrary to this aconstitutive expression pattern was observed in embryos (Sparvoli et al.(1994) Plant Mol. Biol. 24, 743-755).

Stilbene synthase enzymes catalyze the synthesis of stilbenes such asresveratrol or pinosylvin from one molecule of p-cumaroyl-CoA orcinnamoyl-CoA and three units of malonyl-CoA. Resveratrol as well aspinosylvin have photoalexin properties and an antifungal activity, andperform, as phytoalexins in combination with other stilbenes derivedfrom the phenylpropane metabolism, an important function in the defenceagainst pathogens (Hart (1981) Annu. Rev. Phytopathol. 19, 437-458).

STS genes are found in some non-related plant species such as, e.g.,peanut (Schröder et al. (1988) Eur. J. Biochem. 172, 161-169), grapevine(Hain et al. (1993) Nature 361, 153-156) and pine (Fliegmann et al.(1992) Plant Mol. Biol. 18, 489-503) and are organized in larger genefamilies, comprising six or more genes (Lanz et al. (1990) Planta 181,169-175; Wiese et al. (1994) Plant Mol. Biol. 26, 667-677).

Experiments with transgenic tobacco cells indicate that the expressionof the stilbene synthase is regulated mainly at a transcription level,and that the stress-induced signal transduction chain has been preservedin various plant species during the course of evolution (Hain et al.(1990) Plant Mol. Biol.15, 325-335.

STS genes from peanut (Arachis hypogaea) and grapevine (Vitis vinifera)have already been isolated (Schröder et al. (1988) supra or Hain et al.(1993) supra) and expressed in transgenic plants (Hain et al. (1990)supra or Hain et al. (1993) supra).

DNA sequences coding for stilbene synthase are known, e.g., fromEuropean Patent EP 0 309 862, German Patent Application DE-A-41 07 396,European Patent Application 0 464 461, as well as U.S. Pat. No.5,500,367. These documents describe the isolation of stilbene-synthasegenes and their use to produce transgenic plants. The resultingtransgenic plants show greater resistance to various plant pests such asfungi, bacteria, insects, viruses and nematodes. Plasmids containing STSgenes have been deposited with the German Collection of Microorganisms(DSM), Mascheroder Weg 1B, D-38124 Braunschweig. Also included in thedeposition is the VstI-gene from grapevine in the pVstI plasmid, underdeposit number DSM 6002 (DE-A-41 07 396, EP-A-0 464 461, U.S. Pat. No.5,500,367).

While in the meantime also the use of STS coding sequences to producemale, sterile plants and altered blossom colour has been described(German Patent Application DE-A-44 40 200), a possible relation betweenSTS gene expression and ozone induction has remained completelyunexplored up to now.

Meanwhile there are various indications that in order to produce aneffective as possible resistance to disease, based on the expression ofSTS genes in transgenic plants, it is advantageous if the expression ofthe heterologous STS gene (or the heterologous STS genes) in the plantis stimulated first of all by the attacking pathogen, i.e. if it isstimulated first of all by the interaction of plant and pathogen(Fischer and Hain (1994) Current Opinion in Biotechnology 5, 125-130;Fischer (1994) “Optimization of the heterological Expression ofStilbene-synthase Genes for the Protection of Plants”, HohenheimUniversity). This is endorsed particularly by the observation that thepathogen-induced STS gene expression is locally limited to the place ofinfection and is of a transient nature, which means that the STSexpression rises relatively fast to a maximum and declines again in lessthan 48 hours (Hain et al. (1993) supra). Also experiments conducted ontransgenic tobacco plants, in which STS genes were expressed under theconstitutive 35S RNA promotor of cauliflower mosaic virus, showed thatthe resistance to disease achieved in said plants is lower than inplants which expressed the identical genes under the control of thepathogen-inducible homologous STS promotor after fungus infection(Fischer and Hain (1994) supra; Fischer (1994) supra). Anyway, it isdesirable that the STS expression in transgenic, cultivated plantswhich, due to the inserted STS genes show greater resistance to disease,are activated and controlled solely (and not until) by the attack ofpathogens and not additionally (or already before) by undesirableenvironmental stress factors such as ozone.

Thus, it is an important task of biotechnological research of plantprotection to realize a more specific expression of defensive genes inplants, in order to be able to materialize molecular biologicalstrategies for producing plants of greater resistance in an efficientand controllable manner. An important aspect in doing so is to eliminateundesirable, non-specific environmental stimuli such as, e.g., theinduction of certain defensive genes through ozone, ultraviolet light,heavy metals, extreme temperatures and other abiotic stimuli.

Thus, it is an important object of this invention to make available newDNA sequences which play a direct part in the ozone-induced expressionof resistance genes.

Another object of the invention is to show possibilities for removingthe ozone induction, i.e. to eliminate undesirable stimulation of thegene expression through ozone.

Furthermore, an important object of this invention is to provide a DNAsequence with the help of which stilbene-synthase genes can be expressedin transgenic plants only after contact of the plant with the pathogenand not through ozone stimulation.

As mentioned already at the outset, a steady increase in ozone impactcan be observed which also has drastic effects on vegetation. Theobservation and determination of ozone concentrations in the airconstitute already today a focal point of chemical, physical andbiological, environmental research. An important instrument in thiscontext are the so-called biomonitors, with the help of which ozoneimpact and the consequences thereof, particularly phytotoxic effects canbe easily determined both qualitatively and quantitatively.

Thus, a further object of this invention is to provide DNA sequencesthat can be used to produce targeted ozone-inducible promoters. With thehelp of such promotors it is possible for so-called reporter genes, theexpression of which can be proven by simple, enzymatic tests, and whichare well known in biotechnical research, to be used as biomonitors.

Another object of the invention is to provide a system with the help ofwhich certain genes,—whose gene products are able to detoxify oxygenspecies in cells—can be “turned on”, if necessary, as for instance incase of great ozone impact. In other words, by providing DNA sequences,which are responsible for ozone-responsive gene regulation, anozone-inducible expression of said genes such as, e.g., catalase and/orsuperoxide-dismutase genes, shall be rendered possible. Thus, it is anobject of the invention to make available DNA sequences which can beused for producing an ozone-inducible, cellular “ozone protectionsystem”. Further objects of the invention will become apparent as thefollowing description proceeds.

These problems are solved by the subject-matter of the independentclaims, based particularly on the provision of the DNA sequences,according to the invention, which are directly involved in theozone-induced gene expression in plants.

We found, to our surprise, that a certain plant nucleic acid sequence isdirectly involved in the ozone-responsive STS expression. Whileexperiments with transgenic tobacco embryos and plants—which express thecustomary reporter gene uidA from E. coli that codes for aβ-glucuronidase under the control of variously long 5′ deletions of theVstI-promotor from grapevine—indicate that at least a few cis-elements,responsible for the fungus induction, are within the range of thepromotor which comprises base pairs −140 to −280 (calculated from thestart of transcription) (Fischer (1994) supra), the range of the VstIsequence, which comprises base pairs −280 to −430, is essential for astrong activation of the gene expression through ozone. Based on ourexperiments, it was possible to show that a VstI promoter, which is leftwith only base pairs up to and including −280 (and which thus is lackingthe VstI-promoter sequences, located further upstream) is no longerozone inducible. As mentioned above, said shortened promotor isnevertheless still able to indicate pathogen-induced gene expression ofthe coding sequence controlled by same (see Fischer (1994) supra).

Our analyses also lead us to suspect a relation between the treatment ofplants with ozone and an increased biosynthesis or release of ethylenein plant cells. Therefore, an involvement of ethylene-responsiveelements in the ozone-induced gene expression cannot be excluded. Thus,by taking into consideration familiar cis-elements, which are currentlybeing discussed in connection with ethylene-response capacity (Sessa etal. (1995) Plant Mol. Biol. 28, 145-153; Shinshi et al. (1995) PlantMol. 27, 923-932), an involvement of the sequence range of the VstIpromotor, which comprises base pairs −283 to −273, cannot be excluded inan ozone-induced STS-gene expression.

Accordingly, the ozone-responsive DNA sequence range, which is describedhere for the first time, comprises base pairs −270 to −430 of the VstIpromotor from grapevine.

Thus, the present invention relates to the DNA sequence (SEQ ID NO:1),as defined in Claim 1:

ACTTTTCGAG CCCCTTGAAC TGGAAATTAA TACATTTTCC ACTTGACTTT TGAAAAGGAGGCAATCCCAC GGGAGGGAAG CTGCTACCAA CCTTCGTAAT GTTAATGAAA TCAAAGTCACTCAATGTCCG AATTTCAAAC CTCANCAACC CAATAGCCAA T, which is essential forthe ozone-induced gene expression in plants. A preferred version of theDNA sequence, according to the invention, deals with a DNA sequence,which originates from grapevine, and especially preferred from thestilbene synthase gene VstI from grapevine (base pairs −270 to −430).

Furthermore, the invention relates to a promotor region of the VstI-genewhich lacks at least the DNA sequence that comprises base pairs −270 to−430 of the VstI-gene. A preferred version concerns a promotor region ofthe VstI gene, which only comprises the sequence range from the start ofthe translation to base pair −270 of the VstI gene. It is particularlypreferred that the promoter region, which lacks the sequence range −270to −430 of the VstI gene, is able to convey a pathogen-induced geneexpression in plants.

The invention also relates to chimeric nucleic acid molecules, intowhich the DNA sequence of base pairs −270 to −430 of the VstI-gene or atleast a fragment of this sequence range has been inserted. It isespecially preferred that the chimeric nucleic acid molecules, accordingto the invention, render possible, due to the presence of the DNAsequence of base pairs −270 to −430 of the VstI gene or at least afragment thereof, an ozone-inducible expression of the coding regions inplants contained therein.

The nucleic acid molecules can be any nucleic acid molecules, especiallyDNA or RNA molecules, e.g., cDNA, genomic DNA, mRNA, etc. They can benaturally occurring molecules or molecules produced by gene technologyor by chemical synthetic processes.

By making available, according to the invention, DNA sequences, promotorregions, nucleic acid molecules or vectors, it is now possible to mutateplant cells by means of gene technology methods in such a way that theyshow ozone-inducible characteristics. Furthermore, it is now possible tomutate plant cells by means of gene technology methods in such a waythat they characterize one or more genes—which are naturally ozoneinducible, due to the presence of the DNA sequence set out in Claim No.1 or a DNA sequence derivable therefrom or one that is homologous withsaid DNA sequence—as being no longer inducible by ozone but preferablyinducible mainly by pathogens.

A special advantage of the invention is the fact that the ozoneinduction of naturally ozone-inducible genes in plants and plant cellscan be eliminated by deleting the DNA sequence, as set out in Claim No.1, or at least a fragment thereof, in the genes which naturally containthis DNA sequence or a DNA sequence which can be derived therefrom orone that is homologous with said DNA sequence.

Another advantage of the invention is that genes which cannot or cannotsubstantially be naturally induced through ozone, can be characterizedas being ozone inducible by using the invented nucleic acid sequences inplant and plant cells. In a preferred version the nucleic acid sequence,which is responsible for the ozone-inducible expression or at least afragment thereof, controls the expression of genes, the gene products ofwhich are able to detoxify reactive oxygen species that can developamong other things, as a consequence of ozone in plant cells. In aparticularly preferred version, the nucleic acid sequence controls theexpression of catalase and/or superoxide-dismutase genes.

In an alternative version, the DNA sequence that is responsible for theozone-inducible gene expression controls the expression of reportergenes which are measured in order to determine ozone concentrationsquantitatively and/or qualitatively and to evaluate the effects ofozone. Such reporter genes can be, e.g., the uidA gene from E. coli,which codes for the enzyme β-glucuronidase (GUS), luciferase genes orother genes, customary in plant biotechnology. Every expert inbiotechnology, biochemistry or molecular biology is familiar withappropriate reporter genes.

Furthermore, the invention relates to vectors which contain theabove-mentioned DNA sequences or promoter regions or fragments thereof.Thus, this invention relates also to vectors, particularly plasmids,cosmids, viruses, bacteriophages and other vectors, common in genetechnology, which contain the above-mentioned nucleic acid molecules,according to the invention and which, if required, can be used fortransferring said nucleic acid molecules to plants or plant cells.

The invention also relates to transformed microorganisms, such asbacteria, viruses and fungi which contain the nucleic acid sequences,according to the invention.

It is also an object of the invention to provide plants and plant cellswhich are characterized by the absence of the ozone-inducible expressionof genes that are naturally induced in plants through ozone.

This problem is solved by providing the DNA sequence responsible for theozone induction and by making available promoters which lack saidsequence and which for this reason render possible a no longerozone-inducible gene expression of genes in plant and plant cellscontrolled by the promotors.

The problem of rendering possible an ozone-responsive gene expression ofgenes which are not naturally inducible through ozone is likewise solvedby providing the DNA sequence, according to the invention. Thus, plantsand plant cells are made available which in the presence of ozonespecifically define certain characteristics.

A preferred version concerns transformed plants and plant cells in whichgenes are ozone-inducibly expressed, and the gene products thereof areable to detoxify reactive oxygen species in plant cells. Particularpreference is given in this connection to catalase and/orsuperoxide-dismutase genes. As an alternative, it is possible to produceplants and plant cells (including protoplasts) which characterizeso-called reporter genes after induction through ozone and which, ifrequired, can be used as biomonitors.

Thus, the subject-matter of the invention is transgenic plants whichcontain, integrated in the plant genome, the recombined nucleic acidmolecules, as described above. Such plants can, in principle, be anyplants. It concerns preferably a monocotyle or dicotyle useful plant.Examples of monocotyle plants are plants which belong to the genuses ofAvena (oat), Triticum (wheat), Secale (rye), Hordeum (barley), Oryza(rice), Panicum, Pennisetum, Setaria, Sorghum (millet), Zea (corn).Dicotyle, useful plants are, e.g., cotton, leguminous plants such aslegumes, and especially alfalfa, soya bean, rape, tomato, sugar beet,potato, ornamental plants, trees. Other useful plants can befruit-bearing plants (particularly apples, pears, cherries, grapes,citrus fruits, pineapples and bananas), oil palms, tea and cacao shrubs,tobacco, sisal, as well as medicinal plants, such as rauwolfia anddigitalis. Special preference is given to grain, such as wheat, rye,oat, barley, rice, corn and millet, sugar beet, rape, soya, tomato,potato and tobacco.

Furthermore, the subject-matter of the invention is the propagationmaterial of plants, according to the invention, such as, e.g., seeds,fruits, cuttings, tubers, rootstock, etc., as well as constituents ofsuch plants, such as plant cells, protoplasts and calli.

The plant cells include differentiated and non-differentiated plantcells (including protoplasts), as well as plant cells (includingprotoplasts) in which nucleic acid molecules are integrated in the plantgenome or are present as autonomous molecules (including transienttransformation).

In another version, the invention relates to host cells, particularlyprokaryontic or eukaryontic cells, which have been transformed orinoculated with a recombined nucleic acid molecule or a vector, asdescribed above, and cells which originate from said host cells andwhich contain the described nucleic acid molecules or vectors. The hostcells can be bacteria or fungus cells, as well as plant or animal cells.

The object of this invention is also to show methods for the productionof plant and plant cells which are characterized by the lack of anozone-inducible expression of a gene, the expression thereof in plantsand plant cells is naturally stimulated by ozone.

This problem is solved through processes by means of which theproduction of new plants and plant cells, which do not have thisnaturally occurring ozone-induced gene expression, is made possible.

As already mentioned above, it is also the object of this invention toprovide methods for the production of plants and plant cells whichexpress such genes—the expression of which is naturally not, or notsubstantially activated by ozone—after ozone stimulation. This problemis solved by methods with the help of which plants and plant cells canbe produced which, after the invented DNA sequences or at least afragment thereof has been inserted into naturally not ozone-induciblegenes, or genes which are ozone-inducible only to a minor degree expresssuch genes after ozone stimulation.

There are various methods by which such new plants or plant cells can beproduced. For one thing, plants or plant cells can be mutated byconventional gene-technological transformation methods in such a waythat the new nucleic acid molecules are being integrated in the plantgenome, which means that stable transformants are produced.

According to the invention, plants or plant cells which, due to theabsence of the invented nucleic acid sequence or at least a fragmentthereof, no longer show an ozone induction of the gene(s) whichnaturally contains/contain said sequence, are produced by a method whichincludes the following steps:

a) Deletion of the DNA sequence—as defined in Claim No. 1, or a sequencewhich can be derived from said sequence, or which is homologous withsaid sequence, or at least a fragment of such sequence—from a gene whichafter the deletion of the invented DNA sequence, includes regulatoryelements essential for the possibly regulated transcription andtranslation in plant cells, and has at least one coding sequence, aswell as possibly a termination signal for the termination of thetranscription and the addition of a poly-A-tail to the respectivetranscript.

b) Transformation of plant cells with the gene or nucleic acid molecule,produced in step a), and

c) possibly the regeneration of transgenic plants and possibly thepropagation of the plants.

As an alternative it is possible in step a) that instead of deleting thesequence responsible for the ozone-induction, said sequence or at leasta fragment thereof can be inactivated or blocked, e.g., throughmutagenesis, and thus remain in the gene in an inactivated form.Irrespective of the manner in which the ozone-responsive gene range isdeleted, all manipulative measures can be carried out by means ofconventional methods and aids of recombined gene technology (see, e.g.,Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour,N.Y.).

In a particularly preferred version the nucleic acid molecule, which instep b) is transferred to plants or plant cells, contains regulatoryelements which allow, e.g., a pathogen-induced gene expression of thecoding sequence.

According to the invention, plants or plant cells which, due to thepresence of the invented nucleic acid sequence that is essential orco-responsible for an ozone-induced expression of the genes containingsame, or at least a fragment of said sequence, are produced by a methodwhich includes the following steps:

a). Insertion of at least one DNA sequence, according to the invention,which in plants can produce an ozone-induced gene expression, or asequence which is derivable from said sequence, or which is homologouswith said sequence, or at least a fragment of said sequence, in a genewhich is not naturally or not substantially expressed asozone-inducible.

b) Transformation of plant cells by way of the gene or nucleic acidmolecule, produced in step a), which has all elements that are naturallyrequired for the expression in plant cells, and

c) possibly the regeneration of transgenic plants and perhaps thepropagation of plants.

In a preferred version the gene concerned is a catalase dismutase,superoxide-dismutase or a common reporter gene.

Another object of the invention is to show advantageous usage of theinvented nucleic acid sequences.

The invention includes, therefore, uses of the new DNA molecules toproduce the aforementioned plants and plant cells, according to theinvention, which are characterized either by the absence of a certainphenotypical distinguishing mark that is normally influenced by ozone,or which precisely due to the presence of the invented DNA sequencedistinguish themselves from non-transgenic plants and plant cells byozone-induced characteristics.

Furthermore, the invention includes the use of the invented nucleic acidmolecules to produce plants which are characterized by an increasedpathogen-induced but not an ozone-induced resistance to disease.

The invention also relates to the use of the invented nucleic acidsequences or fragments thereof for detecting and identifyingozone-responsive nucleic acid elements.

The expert can identify such ozone-responsive nucleic acid elements byapplying customary molecular biological methods, e.g., hybridizingexperiments or DNA protein-binding studies. For example, as a firststep, the poly (A)⁺ RNA is isolated from a tissue which was treated withozone. Then a cDNA-bank is set up. In a second step and with the help ofcDNA-clones, which are based on poly(A)⁺ RNA molecules from anon-treated tissue, those clones from the first bank are identified byway of hybridizing whose corresponding poly(A)⁺ RNA molecules areinduced strictly in the ozone treatment. With the help of cDNAsidentified in this manner, promoters which have ozone-responsiveelements are subsequently isolated. The nucleic acid sequences andmolecules can be useful instruments when examining and characterizingthese isolated promoters.

The subject-matter of the invention includes also nucleic acid moleculesor fragments thereof which hybridize with one of the above-describednucleic acid molecules or one of the above-mentioned DNA sequences ofthe invention. Within the scope of this invention the term “hybridizing”means hybridizing under conventional hybridizing conditions, preferablyunder stringent conditions, such as are described, for example, inSambrook et al (1989) Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Laboratory Press, Cold Spring Harbor, N.Y.

Nucleic acid molecules which hybridize with the molecules, according tothe invention, can be isolated, e.g., from genomic or cDNA banks.

The identification and isolation of such nucleic acid molecules can beaccomplished by using the nucleic acid molecules, according to theinvention, or fragments of said molecules or the reverse complements ofsuch molecules, e.g., by way of hybridizing according to standardprocedure (see, e.g., Sambrook et al, supra).

Thus, the invention relates also to the use of an invented DNA sequenceor fragments thereof to identify and isolate homologous sequences fromplants or other organisms.

For example, nucleic acid molecules which have exactly or substantiallythe invented nucleotide sequences or fragments of such sequences can beused as a hybridizing probe. The fragments used as a hybridizing probecan also be synthetic fragments which were produced with the help ofcustomary synthesis techniques and the sequence of which basicallycorresponds with that of a nucleic acid molecule, according to theinvention. Once genes that hybridize with the invented nucleic acidsequences have been identified and isolated, it is necessary todetermine the sequence and analyze the properties. To do so, a number ofmolecular biological, biochemical and biotechnological standard methodsare available to the expert.

The molecules that hybridize with the nucleic acid molecules, accordingto the invention, include also fragments, derivatives and allelicvariants of the above-described DNA molecules that contain anozone-responsive sequence in an active or inactivated form, or which arecharacterized by the fact that they no longer have such sequence. Theterm “derivative” means in this context that the sequences of thesemolecules distinguish themselves from the sequences of theabove-described nucleic acid molecules in one or several positions andare to a great extent homologous with said sequences. Homology in thisconnection means a sequence identity of at least 40%, especially anidentity of at least 60%, preferably above 80%, and especiallypreferable above 90%. The deviations from the above-described nucleicacid molecules can have been caused by deletion, addition, substitution,insertion or recombination.

With respect to the nucleic acid molecules which are homologous with theabove-described molecules and which constitute derivatives of suchmolecules, it concerns usually variants of such molecules thatconstitute modifications which perform the same biological function.These may concern naturally occurring variations, e.g., sequences fromother organisms, or mutations in which these modifications can haveoccurred naturally or were introduced through specific mutagenesis.Furthermore, the variations may concern synthetically producedsequences. With respect to the allelic variants, these may occurnaturally as well as be synthetically produced variants, or variantsproduced by recombined DNA methods.

To prepare the insertion of foreign genes into higher plants or into thecells thereof, a large number of cloning vectors are available whichcontain a replication signal for E. coli and a marker gene to selecttransformed bacteria cells. Examples of such vectors are pBR322,pUC-series, M13mp-series, pACYC184, etc. The desired sequence can beinserted into the vector at a suitable restrictive cut. The plasmidobtained is used for the transformation of E. coli cells. Transformed E.coli cells are cultured in a suitable medium and subsequently harvestedand lysed. The plasmid is recovered. Usually restrictive analyses, gelelectrophoreses and other biochemical, molecular biological methods areapplied as an analyzing method to characterize gained plasmid DNA. Aftereach manipulation the plasmid DNA can be split and the gained DNAfragments can be linked with other DNA sequences. Each plasmid-DNAsequence can be cloned in identical or other plasmids.

Many well-known methods are available to introduce DNA into a plant hostcell. The expert can determine, without difficulty, the respectivelysuitable method. These techniques include the following: thetransformation of plant cells with T-DNA by using Agrobacteriumtumefaciens or Agrobacterium rhizogenes as transformation medium, thefusion of protoplasts, the direct gene transfer of isolated DNA inprotoplasts, the electroporation of DNA, the introduction of DNA bymeans of the biolistic method, as well as other possibilities. In doingso, stable, as well as transient tranformants, can be produced.

When injecting and electroporating DNA into plant cells no specificdemands per se is made on the plasmids used. The same applies to directgene transfer. Simple plasmids as, e.g., pUC derivatives, can be used.If, however, whole plants are to be regenerated from cells transformedin this way, the presence of a selectable marker gene is required. Theexpert is familiar with the usual selection markers, and it does notpose a problem for him to select an appropriate marker. Selectionmarkers in common use are those that make the transformed plant cellsresistant to a biocide or an antibiotic, such as kanamycin, G418,bleomycin, hygromycin, methotrexate, glyphosate, streptomycin,sulfonyl-urea, gentamycin or phosphinotricin, etc.

Depending on the method selected for introducing genes into the plantcell, additional DNA sequences may be required. If, for example, the Tior Ri-plasmid is used for the transformation of the plant cell, at leastthe right boundary, but frequently, however, the right and left boundaryof the T-DNA, contained in the Ti and Ri-plasmid must, as flank region,be linked with the genes to be introduced.

If agrobacteria is used for the transformation, the DNA to be introducedmust be cloned in special plasmids, i.e. either in an intermediary or ina binary vector. Due to sequences which are homologous with sequences inthe T-DNA, the intermediary vectors can be integrated in the Ti- orRi-plasmid of the agrobacteria through homologous recombination. Inaddition, the latter includes the vir-region required for the transferof the T-DNA. Intermediary vectors cannot replicate in agrobacteria. Theintermediary vector can be transferred to Agrobacterium tumefaciens(conjugation) by means of a helper plasmid. Binary vectors can replicatein E. coli as well as in agrobacteria. They contain a selection-markergene and a linker or a polylinker which are framed in by the right andleft T-DNA boundary region. They can be directly transformed into theagrobacteria (Holster et al (1978) Molecular and General Genetics 163,181-187). The agrobacterium which serves as host cell shall contain aplasmid that has a vir-region. The vir-region is necessary fortransferring the T-DNA to the plant cell. Additional T-DNA can bepresent. The agrobacterium, transformed in the manner described, is usedfor the transformation of plant cells.

The use of T-DNA for the transformation of plant cells has beenthoroughly studied and is adequately described in EP 120 515; Hoekemain: The Binary Plant Vector System, Offsetdrokkerij Kanters B. V.,Alblasserdam (1985) Chapter V; Fraley et al (1993) Crit. Rev. Plant.Sci., 4, 1-46 and An et al (1985) EMBO J. 4, 277-287.

For the transfer of the DNA to the plant cell, plant explantates can beappropriately cultivated with Agrobacterium tumefaciens or Agrobacteriumrhizogenes. Out of the infected plant material (leaf fragments, stemsegments, roots, but also protoplasts or suspension-cultivated plantcells) whole plants can be regenerated in a suitable medium which cancontain antibiotics or biocides for the selection of transformed cells.The regeneration of plants is carried out according to customaryregeneration methods by using familiar culture media. Plants or plantcells obtained in the above-described manner can then be examined forthe presence of the introduced DNA. Other possibilities for introducingforeign DNA by applying the biolistic method or through protoplasttransformation are known (see, e.g., Willmitzer L. (1993) TransgenicPlants, in: Biotechnology, A Multi-Volume Comprehensive Treatise (H. J.Rehm, G. Reed, A. Pühler, P. Stadler, eds.) Vol. 2, 627-659, V. C. H.Weinheim—New York—Basel—Cambridge).

Although the transformation of dicotyl plants or their cells viaTi-plasmid vector systems and with the help of Agrobacterium tumefaciensis well established, new studies indicate that also monocotyl plants ortheir cells are very receptive to transformation by means ofAgrobacterium-based vectors (Chan et al (1993) Plant Mol. Biol. 22,491-506; Hiei et al (1994) Plant J. 6, 271-282; Deng et al (1990)Science in China 33, 28-34; Wilmink et al (1992) Plant Cell Reports 11,76-80; May et al (1995) Bio/Technology 13, 486-492; Conner and Domiss(1992) Int. J. Plant Sci. 153, 550-555; Ritchie et al (1993) TransgenicRes. 2, 252-265).

Alternative systems for transforming monocotyl plants or their cells aretransformations by means of the biolistic setup (Wan and Lemaux (1994)Plant Physiol. 104, 37-48; Vasil et al (1993) (Bio/Technology 11,1553-1558; Ritala et al (1994) Plant Mol. Biol. 24, 317-325; Spencer etal (1990) Theor. Appl. Genet. 79, 625-631), the protoplasttransformation, the electroporation of partially permeabilized cells andthe introduction of DNA by means of glass fibres.

The transformed cells grow within the plant in the usual manner (seealso McCormick et al (1986) Plant Cell Reports 5, 81-84). The resultingplants can be cultivated normally and grafted with plants which have thesame, transformed genetic trait or other genetic traits. The resultinghybrid individual plants have the respective phenotypical properties.

Two or more generations should be cultivated in order to ensure that thephenotypical characteristic is firmly maintained and propagated. Seedsshould also be harvested in order to ensure that the respectivephenotype or other characteristics are maintained.

By applying the usual methods, transgenic lines can be determined, whichare homozygous for the new nucleic acid molecules and, furthermore,their phenotypical behaviour can be examined for an existing ornon-existing ozone-response capacity and compared to that of hemizygouslines.

Naturally, plant cells which contain the nucleic acid molecules,according to the invention, can also be further cultivated as plantcells, (including protoplasts, calli, suspension cultures, etc.).

Another object of this invention is the use of the nucleic acidmolecules, according to the invention, or fragments of such molecules,or the reverse complements of such molecules to identify and isolatehomologous molecules which include ozone-responsive elements from plantsor other organisms. As to the definition of the term “homology”, pleaserefer to the definition given earlier in the text.

The following examples serve the purpose of explaining the invention.

EXAMPLES Example 1

Construction of the 5′-Deletions of the VstI Promotor as GUS FusionStructure in Binary Vector pPCV002

The 5′ non-coding sequence range of the VstI gene from grapevine, whichhereinafter is referred to as promoter, consists of 1570 base pairs.This promoter, the sequence of which is known from the German PatentApplication DE 41 07 396 and Fischer (1994) supra, was amplified as atemplate with the help of the usual polymerase chain reaction by usingthe following oligonucleotides as primer and the plasmid pVstI,containing the complete VstI gene (DE-A-41 07 396; Fischer (1994)supra):

5′-CCCCAAGCTT CCCCGGATCA CATTTCTATG AGT-3′ (Primer 1, SEQ ID NO: 2)

5′-CGCGGATCCT CAATTGAAGC CATTGATCCT AGCT-3′ (Primer 2, SEQ ID NO: 3).

The PCR was carried out according to the protocol of Perking Elmer(Norwalk, USA) by using the native Taq DNA polymerase by Perkin Elmer.The DNA fragment, amplified under usual PCR conditions, was subsequentlyrecut with the restrictive enzymes HindIII (this restrictive cut isfound at the 5′-end of primer 1) and BamHI (this restrictive cut isfound at the 5′-end of primer 2) and together with theBamHI/EcoRI-fragment from vector pBI101.2 (Jefferson (1987) Plant Mol.Biol. Reporter 5, 387-405), which contains the β-glucuronidase reportergene (GUS, uidA) from E. coli, as well as the termination signal of thenos-gene from A. tumefaciens, subcloned into the pUC18-plasmid(Yanisch-Perron et al. (1985) Gene 33, 103-119; available, e.g., fromBoehringer Mannheim) via the HindIII and EcoRI-restrictive cuts of thepUC18-polylinker. All cloning steps were carried out by using customarymolecular biological methods and aids (for example, Sambrook et al.(1989) supra); restrictive enzymes and other enzymes used for thecloning were acquired from Boehringer Mannheim. The resulting clone(pUC-VstI/GUS) thus contains a translation fusion of the VstI promotorwith the GUS gene, in which the first five STS codons in the readingframe are linked with the GUS gene via a BamHI cut. The sequence rangeof the fusion transition, as well as the sequence of the VstI promotorwere checked for their correctness by means of the enzymatic chain-stopmethod (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467)by using the T7 sequencing kit of Pharmacia (Freiburg).

The cloning of the 5′-deletion mutants in binary vector pPCV002 (Konczand Schell (1986) Mol. Gen. Genet. 204, 383-396) will be explainedbelow. In most cases pUC18-subclones were used as intermediate vectors,because the smaller pUC plasmid is easier to handle in comparison to therelatively large binary vectors.

The plasmid designations result from the approximate length of therespective promotor fragment, calculated from the start of thetranscription, located at a distance of 73 base pairs from the startingcodon (Hain et al. (1993) supra; Fischer (1994) supra). The restrictedcuts used for the gradual shortening of the VstI promotor, arediagrammatically shown in Illustration 1.

p1500GUS: The HindIII/EcoRI fragment, which contains the complete STSpromotor together with the GUS gene, was isolated from the abovedescribed plasmid pUC-VstI/GUS and inserted into the polylinker ofpPCV002, between restrictive cuts HindIII and EcoRI.

p1060GUS: A 1130 Bp-promoter fragment was isolated from pUC-VstI/GUS viarestrictive cuts BamHI and SspI and cloned in BamHI/HincII-linearizedpUC18 (→pUC1130). The GUS gene as BamHI/EcoRI fragment was subsequentlyisolated from pUC-VstI/GUS and cloned between the BamHI cut and theEcoRI cut of pUC1130.

Finally, the fusion was isolated via HindIII/EcoRI double digestion andinserted into the poylinker of pPCV002 via the same cuts.

p930GUS: The 1.0 kb-promotor fragment was isolated from pUC-VstI/GUS viarestrictive enzymes BamHI and HincII and inserted into the polylinker ofpUC18 (→pUC1000), via the same cuts. This was followed by cloninganalogous to p1060GUS.

p740GUS: The approximately 1.6-kb-long HindIII/BamHI promotor fragmentwas isolated from plasmid pUC-VstI/GUS and digested with the restrictiveenzyme DraI. The resulting 810-Bp-long BamHT/DraI fragment wassubsequently cloned in pUC18 via restriction cuts BamHI and HincII(→pUC810). The GUS gene, as a BamHI/EcoRI-fragment, was then isolatedfrom pUC-VstI/GUS and cloned between the BamHi cut and the EcoRI cut ofpUC810. Finally, the fusion was isolated via HindIII/EcoRI doubledigestion and inserted into the polylinker of pPCV002, via the samecuts.

p550GUS: pUC-VstI/GUS was digested with the restrictive enzyme AF1II andthe projecting 5′ ends were filled up with Klenow enzyme (by BoehringerMannheim; dNTPs, likewise from Boehringer Mannheim) according to themanufacturer's instructions. This was followed by re-cutting with therestrictive enzyme BamHI, and the resulting 620 Bp-promotor fragment wasligated in BamHI/HincII linearized pUC18 (→pUC620). Then the cloning wascontinued analogous to that of p1060GUS.

p500GUS: A 570-Bp-long promotor fragment was isolated from pUC-VstI/GUSvia a BamHI/HaeIII double digestion and ligated inBamHI/HincII-linearized pUC19-vector (→pUC570). This was followed byfurther cloning analogous to that for the production of p1060GUS.

p430GUS: Plasmid pUC1000, as described above, was linearized by usingthe restrictive enzyme SnaBI and subsequently digested with HincII. Thelinearized vector was subsequently religated whereby the 500 base pairsof the promotor were deleted between −930 and −430 (→pUC500). Furthercloning was carried out analogous to pUC106GUS.

p280GUS: pUC-VstI/GUS was digested with the restrictive enzyme BanII andthe projecting ends were filled up with Klenow enzyme, and thelinearized vector was subsequently digested with the restriction enzymeBamHI. After isolating the 350-Bp-long blunt end/BamHI-promoterfragment, cloning was continued analogous to p1060GUS.

p140GUS: The above-described pUC subclone pUC620 was digested with therestriction enzymes NsiI and PstI, and the linearized vector wassubsequently cleaned and religated. This resulted in the deletion ofpromotor sequences −550 to −140 (→pUC210). The GUS gene, as aBamHI/EcoRI fragment, was subsequently isolated from pUC-VstI/GUS andcloned between the BamHI cut and the EcoRI cut of pUC1130. Finally, thefusion was isolated via a HindIII/EcoRI double digestion and insertedinto the polylinker of pPCV002 via the same cuts.

p40GUS: The 110 Bp-promoter fragment together with the GUS gene wereisolated from pUC-VstI/GUS by a NheI/EcoRI double digestion, and thefragment was subsequently cloned in XbaI/EcoRI-linearized pPCV002.

pΔGUS: The structure p1500GUS was digested with the restrictive enzymeBamHI, and the cleaned vector was subsequently religated. Thus, theentire promotor fragment was eliminated through the BamHI cut whichvector pPCV002 contains naturally in its multiple cloning spot, besidethe HindIII cut (Koncz and Schell (1986) supra).

P35SGUS: The fusion of the 35S RNA promotor from cauliflower mosaicvirus with the GUS gene, which served as positive control, was isolatedas HindIII fragment from the expression vector pRT99GUS (Töpfer et al.(1988) Nucleic Acid Research 16, 8725) and inserted into the multiplecloning spot of pPCV002.

Example 2

Plant Material, Plant Transformation and Regeneration of TransgenicPlants

Nicotiana tabacum cv. Petit Havana SR1 (Maliga et al. (1973) Nature NewBiol. 244, 29-30) was cultivated as sterile scion culture onhormone-free 1/2 LS-medium (Linsmaier and Skoog (1965) Physiol. Plant18, 100-127) with 1% saccharose at 26° C., 3000 lux and a 16-hourphotoperiod. At 6 to 8-week intervals, sprout segments were transferredto a fresh LS medium. For the transformation of leaf slices withAgrobacterium tumefaciens (Horsch et al. (1985) Science 227 1229-1231) 2to 3-cm-long leaves of sterile scion cultures were stamped into slicesof 1 cm in diameter and incubated for 5 minutes with a suspension ofagrobacteria (approximately 10⁹ cells/mL YEB medium), which containedone of the above-mentioned plasmid structures. The inoculated leafsegments were kept on a hormone-free LS medium for 2 to 3 days at 26° C.During this time, the bacteria overgrew the leaf segments, which weresubsequently washed in liquid LS medium with no hormones and placed onLS-medium with kanamycin (75 μg/mL; Sigma, Munich), cefotaxim (500μL/mL; Hoechst, Frankfurt) and benzylaminopurine (BAP, 0.5 mg/L; Duchef,Haarlem, The Netherlands). After 2 to 3 weeks, transformed sprouts werevisible, which grew roots on hormone-free LS medium with 75 μg/mLkanamycin and 100 μg/mL cefotaxim.

The agrobacteria cultures used for the transformation of thetobacco-leaf segments were produced as follows: First of all theabove-described pPCV002 derivatives were inserted into the E. colimobilizing strain S17-1 (Simon et al (1983) Bio/Technology 1, 784-790).This produced competent S17-1 E. coli cells, as well as thetransformation of the competent cells with the respective plasmidsaccording to Taketo (1988) Biochem. Biophys. Acta 949, 318-324 orHanahan (1983) J. Mol. Biol. 166, 557-580. Subsequently, the E. colistrain, containing the respective binary vector, and the Agrobacteriumtumefaciens-strain GV3101 C58C1 Rif pMP90RK (Koncz and Schell (1986)supra) were cultured in relevant media up to OD₅₈₀=1.0. The followingwere used for the cultivation of the E. coil bacteria: the customary YTmedium (Miller (1972) Experiments in Molecular Genetics, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.); bacto-tryptone 0.8%(w/v), yeast extract 0.5% (w/v), NaCl 0.5% (w/v. pH 7.0) at 37° C. Forthe cultivation of A. tumefaciens bacteria, YEB medium (beef extract0.5% (w/v), yeast extract 0.1% (w/v), bacto-peptone 0.5% (w/v),saccharose 0.5% (w/v), MgSO₄ 2 mM, pH 7.2) were used at 28° C. For thesubsequent conjugation, the bacteria was separated by centrifuging at1500×g for 5 minutes and washed twice in freshly prepared 10 mM MgSO₄solution. Then donor (E. coli) and acceptor (A. tumefaciens) were mixedat a ratio of 1:1 and dripped onto a YEB agar plate. After incubation of16 hours at 28° C., the bacterial drop was rinsed with 10 mM MgSO₄solution and 10⁻², 10⁻³ and 10⁻⁴ dilutions were plated out on YEBselection plates. From a single culture an agrobacterial culture wascultivated at 28° C. which was used for the transformation of N.tabacum.

The transgenic tobacco sprouts, which contained one of theabove-described VstI promotor/GUS fusions respectively, were propagatedvia sprout culture, and partially transferred to soil and cultivated inthe greenhouse under normal conditions (22° C., 60% relative atmospherichumidity, approximately 15,000 lux) up to blooming. The blossoms wereprotected against foreign pollination by means of parchment bags, andripe seed capsules with seeds of the F1-generation were harvested after4 to 6 weeks. For biological tests in the greenhouse, the seed of theF1-generation was laid out on LS medium with 75 μg/mL of kanamycin. Thiswas done, under sterile conditions, directly out of the capsule or aftersterilization of the surface (1. washing briefly with sterile water; 2.incubation for 2 minutes in 70% ethanol; 3. incubation for 10 minutes in3% NaOC1 (13% active chlorine); 4. washing three times with water; 5.drying in the laminar air flow of the safety work bank). Aftercultivation for about two weeks on LS medium with 75 μg/mL kanamycin at25-26° C., 2000-3000 lux and an atmospheric moisture of 60%, it waspossible to clearly distinguish kanamycin-resistant tobacco embryos andkanamycin-sensitive tobacco embryos. Compared to sensitive embryos,resistant embryos showed a pronounced primary root growth. Aside fromthe cotyledons, primary leaves were recognizable after two weeks only inthe resistant embryos. At this “four-leaf stage” the resistantF1-embryos were transferred to soil, hardened and further cultivatedunder appropriate conditions.

Example 3

Plant Cultivation, Ozone-treatment and Harvesting of Leaves

For the subsequent ozone-treatment, transgenic, kanamycin-resistanttobacco embryos were first of all transferred from agar-plates intoflower pots with a 2:1 mixture of standard substrate (typeT/Frühstorfer/Lauterbach) and pearlit and incubated for three weeks in aclimatic chamber with a 12-hour-day/night cycle, 15,000 lux, at aday-time temperature of 25° C. and a night-time temperature of 20° C.,and at 65 to 70% atmospheric humidity and filtered air.

The tobacco plants were subsequently subjected to a single-ozone pulsefor 10 hours and then incubated for 2-14 hours in pollution-free,filtered air. All gassing experiments were conducted under specifiedclimatic conditions, in closed plexiglass boxes which were put into theclimatic chamber. The ozone treatments started always at 9:00 a.m. andextended over the cycle of the day. The ozone was produced by electricdischarge in dry oxygen. The dosing and analyses were carried out undercomputer control (Langebartels et al. (1991) Plant Physiol. 95,882-889). The tobacco leaves of a plant were counted, starting from thetop to the bottom of the plant, and were then classified into leafnumbers 1-10, in which the first leaf was at least 8 cm large. Thetobacco leaves (classified from 1 to 10) were individually frozen inliquid nitrogen at various points in time after the start of theozone-treatment and stored at −80° C.

Example 4

β-Glucuronidase Activity Test

The fluorometric analysis of the GUS-enzyme activity was made strictlyaccording to Jefferson (1987) supra or Jefferson et al. (1987) EMBO J.6, 3901-3907 by using 4-methyl-umbelliferyl-glucuronide (MUG, sigma,Munich). The concentration of the product 4-methylumbelliferon (MU) wasmeasured with a fluorescent photometer (Perkin Elmer LS-2 Bfilterfluorimeter) in a quartz flow cuvette. The GUS activity in plantextracts was calculated in pmol MU×mg⁻¹×min⁻¹. The protein concentrationin the tobacco leaf extracts was determined according to Bradford (1976)Analyt. Biochem. 72, 248-254.

Parallel conducted experiments with ozone-treated grapevine plants andsubsequent Northern Blot analyses showed that gassing with 0.2 μL/L and0.4 μL/L ozone resulted in a substantial induction of the STS-geneexpression on an mRNA plane. For this reason STS genes from grapevineshould be particularly well suited to identify ozone-responsive DNAelements. It should be mentioned at this point that genes which haveshown up to now a significant, reliably measurable ozone induction inozone-treated plants as compared to untreated plants are not available.

In order to be able to analyze the influence of ozone on a promotorplane, F1-tobacco plants (11 weeks old) of the firmly transformedtobacco line, which contains the VstI promotor/GUS fusion structure withthe complete promotor region (p1500GUS), was used in experiments ofozone gassing. GUS-enzyme activities were determined fluorometrically inraw extracts of leaves which were harvested at various times during a10-hour subjection to ozone gassing with various concentrations of ozone(0.1 μL/L ozone, 0.2 μL/L ozone or 0.4 μL/L ozone) and a 14-hourpost-incubation phase in unpolluted air. The results shown inIllustration 2 show a fast ozone-induced increase of the GUS activity ascompared to control plants which were kept in only pollution-free air.Thus, the treatment with 0.1 μL/L of ozone caused an 11-fold induction,and the treatment with 0.2 or 0.4 μL/L of ozone even caused an up to25-fold induction of the expression of the GUS gene, controlled by theSTS promotor, 12 hours after the start of the ozone treatment.

For the identification of cis-regulatory sequences, which areresponsible for the great ozone induction of the STS promotor that hasbeen observed, the transgenic tobacco lines, which contain theabove-described 5′-deletions of the VstI promotor in fusion with thebacterial GUS-reporter genes, were analyzed as independent FO-plants inozone-gassing experiments. The primary regenerated plants werecultivated in sterile culture for several months and propagated viascion culture. Also F1 tobacco plants (11 weeks old) of these stabletransformants were examined for ozone-induced GUS expression.

The transgenic tobacco plants were treated with 0.1 μL/L of ozone for 10hours and subsequently incubated in unpolluted air for another 2 hours.The GUS activity was determined in raw extracts of medium-old leaves andcompared with the enzyme activity in untreated control plants. Theresults of the fluorometric analysis of the GUS-enzyme activities areshown in Table 1. While the GUS activity results in a slight decrease ofthe ozone induction (induction factor drops from about 12 (−1500) toabout 10 (−430) as the promotor range progressively shortens from −1500to −430 in ozone-treated test plants as well as in untreated controlplants, the additional deletion of the promotor range between −430 and−280, produces a drastic reduction of the GUS expression inozone-treated test plants. While plants, in which the GUS gene is underthe control of the −430-5′-deletion promoter, show a 10-foldozone-induction compared to control plants, plants in which the GUS-geneis controlled by the shorter −280-5′-deletion promotor, show only amaximum 2-fold induction of the GUS expression through ozone.Consequently, the promotor range of the VstI gene from grapevine, whichcomprises base pairs −430 to −280, contains cis-active elements whichare essential for a pronounced ozone-induced expression of the STS gene.

These results were confirmed by plant cells which contain theabove-mentioned structures and which were cultivated in plant-cellcultures.

ILLUSTRATIONS AND TABLES Illustration 1

Restriction card of the VstI-promotor/GUS-translation fusion in plasmidpUC-VstI/GUS. The plasmid contains a translation fusion of the VstIpromotor from grapevine with the GUS gene from E. coli. The restrictivecuts used for the cloning of the 5′-deletion mutants have been plotted.nos ter=terminal signal of the nopalinsynthase gene from Agrobacteriumtumefaciens.

Illustration 2

Kinetics of the VstI promotor-controlled induction of the GUS expressionin transgenic F1-tobacco plants, which contain a translation fusion ofthe GUS gene with the complete VstI promoter from grapevine (p1500GUS)through various concentrations of ozone.

The GUS activities were determined fluorometrically in raw extracts oftobacco leaves (leaf No. 4) according to Jefferson (1987), supra. Thevarious harvesting times can be ascertained from the illustration. The10-hour ozone treatment was followed by a 14-hour post-incubation of thetobacco plants in unpolluted air. Control plants were only kept inunpolluted air (without ozone gassing). n=3 or 4; mean values±standarderrors; all tests were carried out as double experiments.

Table 1

GUs-enzyme activities were determined fluorometrically in proteinextracts of medium-old leaves of ozone-treated (+) and untreated (−),independent tobacco transformants.

The transgenic tobacco lines contain various 5′-deletions of theVstI-promotor in fusion with the bacterial GUS-reporter gene.FO-transformants and F1-plants (11 weeks old) were gassed with 100 nL/Lof ozone for 10 hours and subsequently post-incubated for 2 hours inunpolluted air. Mean values±standard errors; all analyses were carriedout as double experiments

Examined, GUS-enzyme activity VstI promotor independent, [pmol MU5′-deleted transgenic, min⁻¹ + mg⁻¹ protein] − induction to positiontobacco lines ozone ozone factor −1500 1F(1)  735 ± 100 63 ± 7 11.7 −7402(F1) 388 ± 59 30 ± 5 12.9 −550 3(F0) 126 ± 13 12 ± 3 10.5 2(F1) 173 ±25 15 ± 3 11.5 −500 5(F0) 148 ± 52 15 ± 6 9.9 −430 6(F0) 141 ± 38 14 ± 410.0 −280 6(F0) 22 ± 4 13 ± 3 1.7 2(F1) 30 ± 3 15 ± 3 2.0 −140 2(F0)  12 ± 0.2  8 ± 3 1.5 3(F1) 24 ± 3 15 ± 3 1.6 −40 3(F1) 24 ± 2 15 ± 31.6 +70 1(F1) 3.5 ± 1  3.5 ± 1  1.0

3 1 161 DNA Vitis vinifera unsure (145) a, c, t, or g 1 acttttcgagccccttgaac tggaaattaa tacattttcc acttgacttt tgaaaaggag 60 gcaatcccacgggagggaag ctgctaccaa ccttcgtaat gttaatgaaa tcaaagtcac 120 tcaatgtccgaatttcaaac ctcancaacc caatagccaa t 161 2 33 DNA Vitis vinifera 2ccccaagctt ccccggatca catttctatg agt 33 3 34 DNA Vitis vinifera 3cgcggatcct caattgaagc cattgatcct agct 34

What is claimed is:
 1. An isolated nucleic acid consisting of thesequence SEQ ID NO: 1, and which conveys an ozone-inducible geneexpression.
 2. The isolated nucleic acid of claim 1, which originatesfrom grapevine (Vitis vinifera).